A recently-developed technique for discovering new drug leads involves the use of nuclear magnetic resonance (NMR) spectroscopy to discover compounds that bind to a particular target molecule such as a protein (see, for example, U.S. Pat. Nos. 5,698,401 and 5,804,390, to Fesik, et al.). The technique involves the determination of a first two-dimensional 15N/1H NMR correlation spectrum of a protein in which nitrogen atom sites have been isotopically enriched with 15N. This first correlation spectrum is obtained for the protein in the absence of any potential ligand compound(s). Next a suspected ligand compound, or a mixture of such putative ligand compounds, is mixed with the isotopically enriched protein, and a second NMR correlation spectrum is obtained. The two spectra are compared, and differences between the two spectra provide information about 1) the existence of binding between any ligand and the host protein, 2) the site(s) of binding, and 3) the strength(s) of binding.
The technique described in Fesik, et al., supra, employs target molecules which have been isotopically enriched with the NMR-detectable 15N spin nucleus. This method relies upon the genetic modification of a suitable microorganism to express the desired protein, protein fragment, or polypeptide, followed by culturing the modified microorganism in a nutrient medium containing assimilable sources of carbon and nitrogen which include 15N-labeled nutrients. Comparatively inexpensive commercially available 15N ammonium salts provided the 15N source.
However, the application of this NMR drug discovery technique to target molecules isotopically enriched with 13C has been hampered by two drawbacks. First, it is comparatively expensive to produce 13C-enriched target molecules in any useful quantities. For example, the production of proteins by genetically modified microorganisms grown in nutrient media containing commercially available uniformly-labeled glucose (glucose-13C6) is expensive. At the time of filing this application, the cost of glucose-13C6 was approximately $480/g. Alternatively, the production of 13C-labeled proteins by including uniformly 13C-labeled amino acids in the nutrient medium is similarly expensive. Second, the biomolecules produced using glucose-13C6 or commercially available uniformly 13C-enriched amino acids are not ideally suited for the NMR correlation spectra technique. Biomolecules expressed by microorganisms grown in nutrient media containing uniformly 13C-enriched starting materials contain adjacent 13C-labeled carbon atoms. Since the NMR technique depends upon detection of spatial spin coupling (i.e., the nuclear Overhauser effect), the relatively strong spin-spin coupling of adjacent 13C nuclei interferes with the desired observation. There is thus a need for the development of site-specifically 13C-enriched amino acids, proteins and polypeptides.